Aus dem Institut für Experimentelle und Klinische

Pharmakologie(Direktor: Prof. Dr. Dr. Ingolf Cascorbi)

im Universitätsklinikum Schleswig-Holstein, Campus Kiel

an der Christian-Albrechts-Universität zu Kiel

Influence of polymorphisms of three TRP genes

on pain sensitivity in neuropathic pain patients

Inauguraldissertation

zur Erlangung der Doktorwürde

der Medizinischen Fakultät der Christian-Albrechts-Universität

zu Kiel

vorgelegt von

Jihong Zhu

aus Hangzhou, Zhejiang, China

Kiel, 2009

1. Berichterstatter: Prof. Dr. Dr. I. Cascorbi

2. Berichterstatter: Prof. Dr. R. Baron

Tag der mündlichen Prüfung 03.11. 2009 zum Druck genehmigt Kiel, den 03.11.2009

gez. Prof. Dr. P. Gohlke

(Vorsitzender der Prüfungskommission)

Table of contents 1 INTRODUCTION 1 1.1 Neuropathic pain overview 1 1.1.1 Definition of neuropathic pain 1 1.1.2 Etiology of neuropathic pain 1 1.1.3 Mechanisms of neuropathic pain 2 1.1.4 Therapy of neuropathic pain 3 1.2 TRP channels and pain 5 1.2.1 TRPV1 5 1.2.2 TRPM8 7 1.2.3 TRPA1 9 1.3 Aim of the study 10 2 MATERIALS AND METHODS 10 2.1 Patients 10 2.2 Volunteers 11 2.3 Experimental pain test measurements 12 2.3.1 Thermal detection, thermal pain thresholds and paradoxical heat

sensation 12

2.3.2 Mechanical detection threshold for modified von Frey filaments 12 2.3.3 Mechanical pain threshold for pinprick stimuli and dynamic

mechanical allodynia 12

2.3.4 Windup ratio-temporal pain summation for repetitive pinprick stimuli

13

2.3.5 Vibration detection threshold and pressure pain threshold 13 2.3.6 Test side and control side 13 2.3.7 Data evaluation 13 2.4. Genotyping 13 2.4.1 Polymerase Chain Reaction (PCR) 14 2.4.2 Pyrosequencing (PSQ) 16 2.5 Statistical analysis 18 3 RESULTS 19 3.1 The characteristics of neuropathic pain in the seven subgroups 19 3.2 The distribution of genetic polymorphisms 24 3.3 Impact of polymorphisms on pain perception 26 4 DISCUSSION 37 4.1 The characteristics of neuropathic pain in the seven subgroups 37 4.2 Neuropathic pain and single nucleotide polymorphism 38 4.3 Genetic variability of TRPV1 and their influence on pain

perception 39

4.4 Association of TRPM8 genotypes with neuropathic pain 42 4.5 Genetic variability of TRPA1 and association to pain perception 43

4.6 Limitation of our study 45 4.7 Conclusion 45 5 SUMMARY 47 6 REFERENCES 49 7 ACKNOWLEDGEMENT 55

Abbreviations:

NMDA N-methyl-Daspartate receptor

CNS central nervous system

BK bradykinin

PGs prostaglandis

5-HT serotonin

PKC protein kinase C

NO nitric oxide

CWT cold withdrawal time

PCR Polymerase Chain Reaction

APS adenosine 5’ phosphosulfate

PNS peripheral nervous system

ALL dynamic mechanical allodynia

WDT warm detection threshold

CDT cold detection threshold

CPT cold pain threshold

WDT warm detection threshold

PHS paradoxical heat sensation

MDT mechanical detection threshold

MPT mechanical pain threshold

TSL thermal sensory limen

CPT cold pain threshold

PHS paradoxical heat sensation

PPT pressure pain threshold

VDT vibration detection threshold

CRPS complex regional pain syndrome

PHN post-herpetic neuralgia

NE norcpinephrinc

COMT catechol-O-methyltransferase

CNS central nerve system

SNP single nucleatide polymorphism

PI(3)K phosphatidylinositol-3-kinase

TRP transient receptor potential channel

DRG dosal root ganglion

TG trigeminal ganglion

NG nodose ganglian

mGluRs metabotropic glutamate receptors

MAPK mitogen-activated protein kinase pathway

NGF nerve growth factor

PSQ Pyrosequencing

GABA gamma-aminobuytric acid

OPRM µ-opioid receptor

TrkA receptor tyrosine kinases

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1. INTRODUCTION

1.1 Neuropathic pain overview

1.1.1 Definition of neuropathic pain

According to the International Association for the Study of Pain (IASP), neuropathic

pain is defined as pain caused by damage of the neural structures that disrupts the

ability of the sensory nerves to transmit correct information to the brain. Therefore,

neurogenic pain syndromes arise as consequence of central and peripheral nerve

damage, which generally characterized by three different properties [1]

(a) it is often experienced in parts of the body that otherwise appear normal,

(b) it is generally chronic, severe and resistant to over-the-counter analgesics,

(c) it is further aggravated by allodynia (touch-evoked pain).

1.1.2 Etiology of neuropathic pain

Neuropathic pain may result from various causes that affect the brain, spinal cord and

peripheral nerve system, including cervical or lumbar radiculopathy, diabetic

neuropathy, cancer-related neuropathic pain, HIV-related neuropathy, spinal cord

injury, trigeminal neuralgia, complex regional pain syndrome type (CRPS) II and

post-herpetic neuralgia (PHN) [2,3]. Based on the location of the nervous system

lesion, neuropathic pain syndromes may be divided into two groups, central and

peripheral. Alterations of central nervous system physiology may play a significant

role in many of the neuropathic pain conditions associated with peripheral pathology,

making the distinction between peripheral and central pain less distinct than once

believed. PHN is a typical example of neuropathic pain with mixed central and

peripheral components [3]. Neuropathic pain, in contrast to nociceptive pain (i.e.

post–operative pain), is described as "burning", "electric", "tingling", and "shooting" in

nature. It can be continuous or paroxysmal in presentation. Neuropathic pain brings

tremendous direct and indirect costs to patients and their families in terms of pain and

suffering, health care expenditures as well as the quality of life.

The hallmarks of neuropathic pain are chronic allodynia and hyperalgesia. Allodynia is

defined as pain resulting from a stimulus that ordinarily does not elicit a painful

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response (i.e. light touch). Hyperalgesia is defined as an increased sensitivity to a

normally painful stimulus [4]. Primary hyperalgesia, caused by sensitization of

C–fibers, occurs immediately within the area of the injury. Secondary hyperalgesia,

caused by sensitization of dorsal horn neurons, occurs in the undamaged area

surrounding the injury. However, clinically, both negative and positive signs may be

present on examination. Negative signs are evidence of nerve damage such as

sensory deficits, weakness, and reflex changes. Positive signs, also indicative of

nerve damage, include hyperalgesia and allodynia. However, the epidemiology of

neuropathic pain has not been adequately studied, partly because of the diversity of

the associated conditions. Demonstrating a lesion of the nervous system compatible

with particular symptoms and signs could provide strong support for considering the

pain to be neuropathic.

1.1.3 Mechanisms of neuropathic pain

With the development of various experimental models of nerve injury, it has become

increasingly clear that both peripheral and central mechanisms could be relevant to

the pathogenesis of neuropathic pain (Figure 1a). Initially, injury or disease affecting

peripheral nerves induces axonopathy and demyelization in injured afferents and

perhaps also in uninjured neighbours, leading to a phenomena termed membrane

remodelling which consequently increases neuronal excitability, and greatly contribute

to the formation of peripheral sensitization. This is due in a large part to

subtype-selective abnormalities in the expression and trafficking of Na+ channels and

perhaps also to altered kinetic properties of unitary channels. The resulting excess

discharge constitutes a primary neuropathic pain signal [5, 6].

Recently, there has been increasing evidence that hypersensitivity and pain that

occurring under various pathological conditions is often due to up-regulated

expression and /or increased sensitivity of transient receptor potential (TRP) channels.

Several lines of evidence point to the involvement of members of TRPV (vanilloid

receptor) family in the aetiology of neuropathic pain [7].

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Sustained painful stimuli result in central sensitization, which is defined as

heightened sensitivity of spinal neurons, reduced activation thresholds and

enhanced responsiveness to synaptic inputs. Under neuropathy status, sustained

ectopic discharge can maintain central sensitization indefinitely and with it “Aβ pain

“and tactile allodynia. At present, it is widely accepted that central sensitization is

largely mediated by the glutamatergic N-methyl-D-aspartate (NMDA) receptor

(Figure 1b). In addition, some evidence has been demonstrated that the

sympathetic nervous system also plays an important role in neuropathic pain by

analgesia following sympathectomy in animals and humans [8, 9]. Furthermore,

several lines of evidence indicated that immune cells, such as endoneurotrophilis

and macrophages, might be involved in the initiation of neuropathic pain and its

maintenance [10, 11].

1.1.4 Therapy of neuropathic pain

The pharmacotherapy is currently the mainstay of treatment in patients with

neuropathic pain. Since hyperexcitability is associated with abnormal sodium channel

regulation in neuropathic pain, alternative treatment strategies have targeted activity

against specific cellular components involved in the pain-processing loop, either by

blocking Na+, Ca2+ and K+ channels as to suppress neuronal excitability at the level of

the spinal cord or by potentiating the effects of the various antinociceptive substances

released via the central descending systems [12].

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Figure 1: (a) Tissue trauma or nerve injury leads to the release of inflammatory mediators as well as increased neuronal excitability at the site of injury, resulting in a reduction in the pain threshold at the site (peripheral hyperalgesia). Central sensitization is an increase in the excitability of spinal neurons as a result of persistent exposure to afferent input from peripheral neurons, CNS = central nervous system, BK = bradykinin, PGs = prostaglandins and 5-HT = serotonin (b) Schematic representation of the activation of NMDA receptor in relation to pain. The activation of NMDA in response to the released glutamine results in the removal of Mg2+ which leads to the ca2+ influx; ca2+ ions flowing into cell activates the protein kinase C (PKC), an enzyme required for the synthetics of NO, followed by the activation of substance P that binds to the NK-1 receptor triggering c-fos gene expression and hypersensitivity.

A wide variety of drugs, such as 5% lidocaine patches, nonsteroidal anti-inflammatory

drugs (NSAIDs), anticonvulsants (e.g. carbamazepine, gabapentin, lamotrigine), and

tricyclic antidepressants (TCAs), e.g. amitriptyline hydrochloride, desipramine

hydrochloride), could be used as first-line therapy in the treatment of neuropathic

pain. The second-line drug, which mainly encompasses narcotics analgesics or

NMDA receptor antagonist, could be applied to patients who do not respond to

treatment with first line agents. The choice of medication should be directed toward

the type of painful symptom described. Patients who do not respond to monotherapy

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with any of the first- or second-line agents may respond to combination therapy or

may be candidates for invasive therapy in pain clinic [13, 14].

Nowadays, pain research is directing on new molecular methods, such as gene

therapy, stem cell therapy and viral vectors for delivery of biologic antinociceptive

molecules. These methods could provide a new therapeutic approach to neuropathic

pain relief. Pharmacological treatment for the symptoms of painful neuropathy,

however, is still considered to be difficult, as the treatment effect is frequently variable

between individuals. Traditionally, this variation has been explained by variable

bioavailability, differences in intensities of pain stimuli and individual differences in

pain perception. Recently, there is increasing evidence supporting that

polymorphisms in some candidate genes may affect the expression and function in

target product, leading to variability in pain perception or dose requirement of drugs

used for pain treatment [15,16].

1.2 TRP channels and pain

Transient receptor potential (TRP) channels have been found in many cell types.

Three TRP channels including TRPV1, TRPM8 and TRPA1, which have been shown

to be expressed in primary afferent nociceptors, are gradually emerging as sensory

transducers that may participate in the generation of pain sensations evoked by

chemical, thermal and mechanical stimuli (as shown in Figure 2).

1.2.1 TRPV1 Since the molecular identification of the capsaicin receptor, now known as TRPV1,

transient receptor potential (TRP) channels have occupied an important place in the

understanding of sensory nerve function in the context of pain. Functional TRPV1 is

expressed both in different level of neuron tissues like brain, dorsal root ganglion

(DRG), trigeminal ganglion (TG), nodose ganglian (NG) neurons and in non-neuronal

tissues which include gastric epithelial cells, airway epithelial cells, liver, vascular

endothelium, mast cells and bladder. The increase in TRPV1 expression was

suggested to contribute to the pathogenesis of various disease states, such as

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multiple types of pain, vulvar allodynia as well as inflammatory bowel disease,

pancreatitis and migraine [17, 18]. TRPV1 function has also been investigated in

models of neuropathic pain. Several lines

Figure2. Selected stimuli and intracellular pathways that contribute to the sensitization of TRP function in terminal primary sensory neurons. Besides activation by endogenous activating lipids via PLC and PLA pathways, the TRPV1 receptor can be activated by heat (>43 °C), acid and pungent vanilloid compounds. In contrast to the TRPV1 receptor, The TRPM8 receptor can be activated by many cooling compounds (23 °C-26 °C) and odorants like menthol. In addition, the TRPA1 receptor can be activated by endogenous inflammatory mediators and by diverse exogenous irritants, including mustard oil, garlic and allicin. Probably, TRPA1 is functionally coupled to TRPV1 and regulated by similar PLC-dependent sensitization pathways. However, whether the TRPA1 receptor respond to mechanical stimuli is unidentified. BK = bradykinin, PGs = prostaglandins and 5-HT = serotonin, PLA/PLC pathway = phospholipase A / phospholipase C, PGE2 = Prostaglandin E2, AA = arachidonic acid, GPCR = G-protein-coupled receptor, 5HT-2A = serotonin receptor. of evidence point to the involvement of members of the TRPV family, especially

TRPV1 in the aetiology of neuropathic pain [19]. It can be speculated that

up-regulated expression of TRPV1 in myelinated fibers (A-neurons) is part of the

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neuropathic pain phenotypic switch and contributes to hyperalgesia. TRPV1 also

plays an important role in chemical and thermal hyperalgesia in a model of diabetic

neuropathy. The application of Trpv1-specific siRNA VsiR1 for rats with neuropathic

pain showed that the Trpv1-specific siRNA VsiR1 diminishes the frequency of

cold-induced paw lifting by approximately 50%, indicating a reduction of cold allodynia

[18]. More interestingly, animal experiment showed this receptor may have a

protective role in the development of mechanical hyperalgesia [20].

The TRPV1 channel is encoded by the TRPV1 gene, located on chromosome

17p13.3. Several exonic TRPV1 polymorphisms in coding region have identified

recently, including the two common single nucleotide polymorphism (SNP) rs222747

and rs8065080, also known as TRPV1 Met315lle and IIe585Val, which substitutes

methionine to isoleucine at codon 315 and isoleucine to valine at codon 585,

respectively. To date, there have been few studies in the literature reporting the

association between genomic variations of the TRPV1 and the pain perception or the

development of the pathologic pain in humans. An in vitro study by Kayes et al [21]

failed to confirm that the IIe585Val substitution alters the receptor function. Another

study by Kim et al, however, suggested that this substitution at codon 585 significantly

influences the cold withdrawal time (CWT) in Caucasian females [20]. Haplotypes

analysis including these two polymorphisms were recently identified by Kim et al in a

European Americans, but there was no significant association between short duration

cold pain sensitivity and variations in TRPV1 in a gender dependent manner [22]. Until

now, the detailed mechanism explaining how these SNPs influence the mRNA

expression and their relevant function in vivo is unclear. In addition, it remains open

whether these polymorphisms in human could influence pain perception on the

condition of neuropathic pain. Hence, further investigation to explore these

associations between polymorphism and pain perception is necessary.

1.2.2 TRPM8

The TRPM8 channel is encoded by TRPM8, which is located on the chromosome

2q37.1. TRPM8 is expressed in prostate and in small-diameter trigeminal and dorsal

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root ganglion (DRG) neurons, suggesting its specific expression in C-and possibly

A-fibers, where TRPM8 functions as cold sensing pain pathway and sensor [20, 23].

TRPM8, also known as the menthol receptor, can be activated by many cooling

compounds and odorants, which are dependent on different factors such as intra and

extracellular Ca2+ concentration and PH [19, 20]. It has been previously proposed that

TRPM8 is a candidate as sensory transducer contributing to pain hypersensitivity due

to its property of responding to both innocuous and noxious temperature. Recently, it

has been shown that ethanol is capable of inhibiting this channel as one of known

TRPM8 modulators, but the exact mechanism how the ethanol affect or modulate the

activity of TRPM8 channel is still poorly understood.

More interestingly, it has been demonstrated that activation of TRPM8 in sensory

afferents of neuropathic pain rat models by either cutaneous or intrathecal application

of pharmacological agent or modest cooling causes inhibition of sensitized pain

response, thereby inhibiting the characteristic sensitization of dorsal-horn neurons

and behavioural-reflex facilitation. The mechanism of analgesic effect in TRPM8

activation could be due to the central mediation and possibly relies on group II/III

metabotropic glutamate receptors (mGluRs), which very likely respond to glutamate

released form TRPM8-containing neurons to suppress nociceptive inputs [24].

There is increasing evidence that mutation in coding regions could alter the function of

the TRPM8 channel. Colburn et al showed that sensory neurons derived from trpm8

null mice lack detectable levels of TRPM8 mRNA and protein and that the number of

these neurons responding to cold (18°C) and menthol (100 mM) is greatly decreased.

Furthermore, compared with WT mice, null mice display deficiencies in certain

behaviours; these results suggest that TRPM8 may play an important role in certain

types of cold-induced pain [25]. However, in a study in humans, Kim et al could not

find any association between TRPM8 and cold or heat pain sensitivity and speculated

that TRPM8 receptor was possibly not activated by cold temperature (2-4°C) used

[22].

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1.2.3 TRPA1

The TRPA1 protein encoded by the TRPA1 gene, is located on the chromosome 8q13.

As a member of the TRP family of ion channels, TRPA1 is mainly expressed in the

inner ear, trigeminal and DRG neurons, where there have been proposed roles in

sensing sound, painful cold, and irritating chemicals [19].

Traditionally, the TRPA1 was proposed as a candidate for the hair-cell transduction

channel, but Kwan and colleagues found that TRPA1 seems to be unnecessary for

hair-cell transduction [26]. The observation of trpa1−/−(lacking trpa1) mice revealed

that neither hearing behaviour nor transduction currents changed in response to

bundle deflections, further confirming the possibility that another TRP channel can

compensate the loss of TRPA1.

The majority of studies point to an important role for TRPA1 in the pain response to

endogenous inflammatory mediators and to diverse exogenous irritants, including

mustard oil, garlic, wintergreen oil, clove oil, ginger and cinnamon oil, all of which elicit

acute painful burning or pricking sensation [20]. Some behavioural studies in mice

lacking trpa1 confirmed these effects in nociception. More interestingly, being

consistent with a role in nociception, TRPA1 is highly co-expressed with TRPV1 in

small-diameter peptidergic nociceptors while it is rarely co-expressed with TRPM8.

TRPA1 has also been linked to cold hyperalgesia in the condition of neuropathic pain.

Both inflammation and nerve injury increase the expression of TRPA1 in DRG

neurons. Such over-expression, which appeared to be driven by nerve growth factor

(NGF)-engaging the p38mitogen-activated protein kinase pathway (MAPK),

contributes to injury-induced cold hyperalgesia because trpa1 knock down by siRNA

strategies suppressed cold hyperalgesia in a rat nerve injury model. Obata et al

further confirmed that NGF-induced TRPA1 increase in sensory neurons via p38

activation is necessary for cold hyperalgesia [27]. Therefore, blocking TRPA1 in

sensory neurons might provide a fruitful strategy for treating cold hyperalgesia caused

by inflammation and nerve damage.

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From some animal experiments, it has been obviously shown that mice trpa1 gene

knock out or trpa1 antagonist exhibited behavioural change in response to the pain

stimuli, which illustrated the important role of TRPA1 in pain perception. Genomic

variations may alter protein expression or possibly have significant consequences on

the function of the protein. Up to date, there have been few data suggesting that

polymorphism in the trpa1 gene might contribute to the interindividual variation in pain

perception. For example, in the study by Kim et al [22], it could be demonstrated that

homozygote carriers of the intronic TRPA1 G38218A variant (rs1198795) have less

pain tolerance to the cold stimuli compared to homozygote G carriers. But detailed

knowledge of regulation is less available. Hence, more research work intended to

clarify clinical impact of these genetic variations is required.

1.3 Aim of the study

Candidate gene studies on the basis of biological hypotheses have been a practical

approach to identify relevant genetic variation in complex traits. There is growing

evidence showing that single nucleotide polymorphisms (SNPs) in candidate genes,

including TRPV1, TRPA1 and TRPM8, may influence pain sensitivity in animal

models of neuropathic pain. However, the exact role of these SNPs in pain perception,

interpretation and behavioural expression in humans are currently unknown. As we

know, the research for predictive SNPs within genes may require the examination not

only of the exons, but also of the promoters and/or the area of intron-exons

boundaries and mRNA processing signals. Therefore, in this study, two SNPs were

selected from TRPV1, 3 SNPs from TRPA1 and 6 SNPs from TRPM8. The SNPs

were located in exonic and promoter regions and were examined for their frequency in

a large sample of patients with different neuropathic pain entities and healthy controls.

They were further investigated for their effects on pain sensitivity.

2 MATERIALS AND METHODS

2.1 Patients

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The study was carried out in cooperation with the German Research Network on

Neuropathic Pain headed by Prof. Dr. Ralf Baron, Division of Neurologic Pain

Research, Department of Neurology at the University Hospital Schleswig-Holstein

Campus Kiel and Prof. Dr. Dr. Thomas Toelle, Department of Neurology at University

Medical Centre Rechts der Isar of the Technical University Munich. The study was

performed in accordance to the principles of the Helsinki declaration. All patients gave

their written informed consent and the ethics committee of the medical faculty of the

University of Kiel and the local ethics committees of the participating centres

approved the study. We analyzed the neuropathic pain patient population in a primary

study. All patients were Caucasians and unrelated to each other. So, a total of 296

patients were recruited to the current study for genotyping. Due to the failed collection

of clinical data in some cases, there were 236 patients with a mean age of 57.4 years

(140 women, 96 men) investigated by further statistic analysis (Tab. 1).

Table 1: Demographic data of 236 neuropathic pain patients

Subgroup number (male/female) Age (years)

Central pain 9/8 51.1 ± 11.7 Ccomplex regional pain syndrome 9/47 51.1 ± 13.3 Peripheral nerve injury 13/14 52.6 ± 15.3 Post-herpetic neuralgia 5/22 52.2 ± 16.7 Polyneuropathy 42/29 67.4 ± 12.0 Trigeminal pain 13/17 60.9 ± 12.7 Other neuropathy 5/2 56.5 ± 15.7

Data expressed as the number of patients, mean ± SD

2.2 Volunteers

For comparison of the genotype distribution, 252 healthy volunteers were enrolled at

the Institute of Pharmacology of the Kiel University under a clinical protocol approved

by ethics committee of the medical faculty of the University of Kiel. All volunteers gave

their written informed consent and were German Caucasians.

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2.3 Experimental pain test measurements

In each participating centre, all 13 QST procedures were performed by trained

observers using the same equipment and standardized instructions to the patients. All

test techniques are based on the quantitative sensory testing of the German

Research Network on Neuropathic Pain (DFNS) [28, 29].

2.3.1 Thermal detection, thermal pain thresholds and paradoxical heat

sensation

The thermal test includes CDT (cold detection threshold), WDT (warm detection

threshold) , PHS (paradoxical heat sensation), TSL (thermal sensitivity limen) and

CPT (cold pain threshold). Cold and warm detection thresholds were firstly measured.

In addition, patients were asked about paradoxical heat sensations (PHS) during the

thermal sensory limen (TSL) procedure of alternating three times of warm and cold

stimuli. Then cold pain and heat pain thresholds were determined (CPT, HPT).

2.3.2 Mechanical detection threshold for modified von Frey filaments

The mechanical detection threshold (MDT) was measured with a standardized set of

modified von Frey hairs that exert forces upon bending between 0.25 and 512 mN.

Five threshold determinations were made by using the “methods of limits”, the final

threshold was the geometric mean of these five series. Similarly, the mechanical pain

threshold (MPT) was measured using custom-made weighted pinprick stimuli as a set

of seven pinprick mechanical stimulators, which usually were applied at a rate of 2 s

on, 2 s off in an ascending order until the fist percept of sharpness was reached.

2.3.3 Mechanical pain threshold for pinprick stimuli and dynamic mechanical

allodynia

Mechanical pain sensitivity (MPS) was assessed using the same set of pinprick

stimuli to obtain a stimulus-response function for pinprick-evoked pain. Patients were

asked to give a pain rating for each stimulus on a “0-100” numerical rating scale.

Furthermore, dynamic mechanical allodynia (ALL) was assessed as part of the test

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above, using a set of three light tactile stimulators (cotton wisp, cotton wool and brush)

as moving innocuous stimuli.

2.3.4. Windup ratio-temporal pain summation for repetitive pinprick stimuli

In this test, after a single pinprick stimulus and a series of 10 repetitive pinprick stimuli

of the same physical intensity separately applied to the patients, patients were asked

to give a pain rating representing the single stimuli, and the estimated mean over the

whole series of 10 stimuli using a “0-100” numerical rating scale. Wind-up ration

(WUR) was calculated as the ratio: mean rating of the five series divided by the mean

rating of the five single stimuli.

2.3.5 Vibration detection threshold and pressure pain threshold

The vibration detection threshold (VDT) was recorded at a time point when patients

could not feel the vibration of tuning fork placed over a bony prominence. VDT was

determined as a disappearance threshold with three stimulus repetitions. In addition,

the Pressure pain threshold (PPT) was measured by using a pressure gauge device.

The PPT was determined with three series of ascending stimulus intensities, each

applied as a slowly increasing ramp of 50 kPa/s (~5 kg/cm2 s).

2.3.6 Test side and control side

This test stimuli were applied in runs alternating between the affected test side and a

contralateral non-afftected control site.

2.3.7 Data evaluation

In order to describe which measure is more sensitive to detect sensory plus and

minus signs, the absolute reference data were used to normalize test results of

individual patients by calculating the z-transform: Z=(valuepatient–meancontrols)/SDcontrols.

Subjects were blinded with regard to the temperature of the stimulus.

2.4 Genotyping

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To study possible association between neuropathic pain and single nucleotide

polymorphism (SNP) in candidate genes (Table1), a total of 11 single nucleotide

polymorphism in 296 patients and in 253 volunteers was genotyped by PSQ,

respectively. Besides volunteers, prepared DNA was obtained from Department of

Neurology of the Munich University Medical Centre and all DNA sample stored at -4

°C until PCR reactions were performed. Primer and pyrosequencing primer were

designed by PSQ assay design software (Biotage, Uppsala, Sweden)

2.4.1 Polymerase Chain Reaction (PCR)

DNA fragment containing polymorphic sites were amplified from genomic DNA using

specific primers (as showed in Tab. 2), which were designed by pyrosequencing PSQ

assay design software (Biotage, Uppsala, Sweden). DNA amplifications for these

SNPs were typically performed under similar conditions for each reaction with 1µl of

genomic DNA, 0.2 µM of forward and reverse primers (TIB Molbiol, Berlin, Germany),

2.5 µM magnesium chloride, 1.0 µM dNTPs (Biotage) and 0.15 µl of Taq DNA

polymerase (Invitrogen, Karlsruhe, Germany) , in a total volume of 25 µl (Tab. 3). PCR

was performed under the following conditions: initial denaturation for 4 min at 94 °C,

followed by 50 cycles at 94 °C for 30 s, annealing at 63 °C (61 °C for SNP rs13268757)

for 30s and at 72 °C for 30 s, and 7 min at 72 °C (Tab. 4). All PCR amplifications were

carried out using a GeneAmp 9700 thermocycler (Applied Biosystems, Darmstadt,

Germany). For controlling the PCR reaction, the DNA fragments were separated on a

2.0% agarose gel (AppliChem, Darmstadt, Germany) and visualized after ethidium

bromide staining by a digital image station (Kodak, Stuttgart, Germany).

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Table 2: Primers used for genotyping of polymorphisms in three TRP genes Gene SNP ID mRNA

location

Primer name

Primer sequence

TRPV1 rs222747 G1103C TRPV1-1 F:5'ACG AAG TTT GTG ACG AGC ATG TA

R:5'Bio-TCC CTT CTT GTT GGT GAG CT

S:5'-ATG TAC AAT GAG ATT CTG AT

rs8065080 A1911G TRPV1-2 F:5'-Bio-GCG CTG ACC AAG CTC ATT ACC T

R:5'-TGG CCT CGG CTC TGA TGA

S:5'-CCG TTT CAT GTT TGT CTA

TRPM8 rs17862932 C2235T TRPM8-4 F:5'-GGG TCC AGG AAG AAA CCT GTC

R:5'-Bio-GCG ATC TAG AAG ACC ACA TTC C

S:5'-GTA CTA TGTTGT GGT GTT CTT

rs7593557 G1296A TRPM8-3 F:5'-Bio-GAG TCT TTT CCT GCC CTC

R:5'-CGG TCA TTG GTG AAA ATC TCA T

S:5'-CAG TTA TCC TTG TCT TGC

rs13004520 G780C F:5'-TTT AGC CCA GTA CCT TAT GGA TG

rs28901637

rs11562975

A787T

G790C

TRPM8-1

R:5'-Bio-CTA GCT GAT TCC GGA GCT TTG

rs17868387 A792G S:5'-TTA TGG ATG ACT TCA CAA

TRPA1 rs13268757 C182T TRPA1-1 F:5'-Bio-CTC CAG GGC GCC ACA TCT

R:5'-TTT CGC TGC CTG TGA GCT G

S:5'-GGT GGG GTC AAT GAA

rs920829 G710A TRPA1-2 F:5'-TGA TCA TTG CGT GCA CCA C

R:5'-Bio-TCC TCT TAA GCG GGG AGT ACA TAC

S:5'-CGT GCA CCA CAA ATA AT

rs959976 A3228G TRPA1-3 F:5'-Bio-TTG TCT TAT TTC CCC AGT GCA A

R:5'-TGG CAC ATG CTT AAG AAA AAG TTC

S:5'-ATT TTT TAT CCG ACA GC

Abbreviations: F, forward primer; R, reverse primer; S, sequencing primer; dNTP, deoxynucleotide triphosphates;

Dispensing order: sequence order given by Pyrosequencing software according to each analyzed template

sequence.

Formatierte Tabelle

- 16 -

Table 3: PCR reaction reagents used for amplification of SNPs in three TRP genes.

Reagent Volume [µl] Concentration

Taq-buffer (10x) 2.5 1 x MgCl2 (50 mM) 1.25 2.5 mM dNTPs (10 mM) 2.5 1 mM Primer forward (10 mM) 0.5 200 nM Primer reverse (10 mM) 0.5 200 nM Taq-DNA-polymerase (5U/µl)

0.15 0.03 U/µl

DNA (30-100 ng/µl) 1.0 H2O 16.9 Total volume 25.0

Table 4: Thermocycler conditions of PCR-reaction used for amplification of SNPs in three TRP genes.

Denaturation Denaturation Annealing Elongation Terminal elongation

4 min 30 s 30 s 30 s 7 min 94 °C 94 °C 63 °C* 72 °C 72 °C

45 cycles * 61 °C for SNP rs13268757

2.4.2 Pyrosequencing (PSQ)

10 µl of PCR-reaction product were aliquoted in each well with 30 µl of pure water

(Biochrom AG; Berlin, Germany) and 37 µl of binding buffer (Biotage) as well as 3µl

streptavidine-coated sepharose beads (GE Healthcare Bio-Sciences, Uppsala,

Sweden), the mixture were incubated in a shaker for 20 min. Afterwards using the

Vacuum Prep workstation, biotinylated single-stranded PCR fragments were treated

with 70% ethanol (Merck, Darmstadt, Germany), 0.1%mol/l NaOH and washing buffer

(Biotage),and transferred to 96-well plates, containing annealing buffer and 10 pmol of

sequencing primer in a total volume of 12 µl each. Hybridization was applied by

incubation at 80 °C for 2 min, the plate was cooled to room temperature.

- 17 -

According to the volume information, the enzymes, which include DNA polymerase,

ATP sulfurylase, luciferase and apyrase, the substrates including adenosine 5’

phosphosulfate (APS) and luciferin, as well as the nucleotides were filled in the tips

stored in a dispensing tip holder, from where they were added automatically into each

well of the 96 wells plate containing the sequencing primer /PCR product mix.

The pyrosequencing technique is based on the detection of pyrophosphate released

during polymerization of DNA as illustrated in Figure 3.

Figure 3a: Step 1. A polymerase catalyzes incorporation of nucleotide acid chain. As result of

the incorporation, a pyrophosphate molecule (ppi) is released and subsequently converted to

ATP by ATP sulfurylase.

Figure 3b: Step 2. Meanwhile, light is produced in the luciferin reaction during which a

luciferin molecule is oxidized. The light is proportional to the amount of incorporated

nucleotide and seen as a peak in the program

- 18 -

Figure 3: Step 3. The excess of the added nucleotide wil be degraded by apyrase, the

produced ATP also will rapidly be degraded by apyrase.

Figure 3c: Step 4. Pyrosequencing raw data obtained as pyrogramme, in which proportional

peak signals represent one or two base incorporations. Nucleotide addition, according to the

dispending order, is indicated below the program and the obtained sequence in indicated

above the program.

2.5 Statistical analysis

Descriptive data are presented as mean±SD. The Kruskal-Wallis test and the

Jonckheere-Terpstra test were performed to test the association between SNPs and

13 pain test parameters, using computer software SPSS (version 11.5). A

Kruskal-Wallis test was used to determine whether continuously measured QST

parameters differed according to variants location, and to determine the association

between the candidate SNPs and the QST parameters. The Jonckheere-Terpstra test

was used to assess the statistical significance of the trend in continuously measured

- 19 -

QST parameters across genotypes. In addition, Mann-Whitney U-test (multiple

comparison) was used to compare the difference dependent on genotype either on

test side or on control side. To exam the equality of the allele and genotype

frequencies between the patients and controls, Statcalc (version 6) was used for

calculation of odds ratios. Haplotype analysis was performed using the SHEsis

software, which provided free service from online. A P-value below 0.05 was

considered to be statistical significant and calculated as two-sided significance.

3. RESULTS

3.1. The characteristics of seven subgroups of neuropathic pain

Mean values of all quantitative sensory testing (QST) parameters of test and control

sides are shown in Figure 4. These QST parameters were objectively designed to

detect pain patterns after partial nerve lesion, which is indirectly consistent with the

characteristics of different afferent nerve fibers (nociceptive C-fibers and

non-nociceptive myelinated A-fibers). Theoretically, cutaneous sensitivity is mediated

by various populations of Aβ, Aδ, and C-fiber afferents. Brush and touch by a blunt

von Frey type probe predominately activate Aβ-fiber low–threshold mechanoreceptors.

Gently cooling the skin activates Aδ-fiber thermoreceptors. Contact with sharp objects

such as pinpricks predominantly activates Aδ-fiber nociceptors. Gently warming the

skin activates C-fiber thermoreceptors. Painful heat stimuli activate both Aδ and

C-fiber nociceptors, but due to their lower thresholds only C-fibers are involved in heat

pain threshold. Therefore, based on the quantitative sensory testing (QST), the

Aβ-fiber can be represented by VDT and MDT, whereas the C-fiber function is

represented by the WDT and HPT. The presence of abnormal cold pain threshold

(CPT) indicates a disturbance of Aδ-cold sensation.

The normal range from plus 2 to minus 2 in patients profile was presented by grey

zone. The minus z-score represents loss of sensory function, those minus z-score

outside of normal range shows significant loss of sensory function such as

hypoesthesia. In contrast, the plus mean z-score above normal range represents the

- 20 -

gain of sensory function like hyperalgesia or allodynia.

Figure 4: Z- score sensory profiles of 235 patients on both test and control sides (test side represented the affected side of neuropathic pain, whereas the control side signified the non-affected side of neuropathic pain). These patients, who are being scattered among seven types of neuropathic pain, exhibited different response to 11 items of QST parameters applied. Since the majority of mean value of z-score was distributed within the normal range, the patients were subsequently divided into subgroups for further investigation.

To further illustrate the characteristics of subgroups of neuropathic pain, patients were

divided into seven subgroups, including central pain, complex regional pain

syndrome(CRPS), peripheral nerve injury, polyneuropathy, trigeminal pain, post

herpetic neuralgia and other neuropathy. The comparison data on both test and

control side among subgroups are shown in Figures 5 a-g. Compared to the control

side, central pain patients apparently exhibited hypoesthesia in CDT, WDT, TSL MDT

and VDT on test side (Figure 5a). Similarly, hypoesthesia with regard to MDT and

VDT was also observed in post herpetic neuralgia (PHN) (Figure 5e), polyneuropathy

(Figure 5f) and peripheral nerve injury patients (Figure 5d), respectively. On the

- 21 -

contrary, the CRPS patients showed static hyperalgesia to blunt pressure (PPT) on

the test side (Figure 5b). In the trigeminal pain (Figure 5g) and other neuropathy

groups (Figure 5c), there is no obvious loss or gain of sensory function by comparing

test and control side.

Figure.5a. Pain patterns of test and control side in central pain patients. Compared to the control side, central pain patients apparently exhibit hypoesthesia to thermal stimuli (CDT, WDT, TSL) and VDT at test side.

Figure 5b: Pain patterns of test and control side in complex regional pain syndrome (CPRS) patients. QST-profile of test side shows static hyperalgesia to blunt pressure (PPT). There was neither allodynia nor hyperalgesia on the control side.

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Figure 5c: Pain patterns of test and control side in other neuropathy subgroup. Neither hypralgesia nor alodynia was observed on test side and control site.

Figure 5d: Pain patterns of test and control side in peripheral nerve injury patient. QST-profile of the test side referred to the affected side confirms hypoesthesia in vibration detection threshold (VDT).

- 23 -

Figure 5e. Pain patterns of test and control side in post-herpetic neuralgia (PHN) patient, Comparing the control side, the mechanical detection threshold (MDT) of test side decreased dramatically, exhibiting tactile hypoesthesia.

Figure 5f: Pain patterns of test and control side in polyneuropathy patients. QST-profile of test side compared to the control side suggests tactile hypoesthesia (MDT) and hypoesthesia in vibration

detection threshold (VDT), even though the pattern of control side is more close and similar to the test side.

- 24 -

Figure 5g: Pain patterns of test and control side in trigeminal pain patients. QST-profile of the control side versus test side shows no significant difference.

3.2. The distribution of genetic polymorphisms

The genotype distribution of all investigated polymorphisms showed no deviations

from Hardy-Weinberg equilibrium neither in patients nor in controls. To evaluate

whether the genotypes investigated may act itself as susceptibility factors for

neuropathic pain, the genotype and allele frequencies were compared between the

case group and healthy volunteers. For most genotypes and alleles, there was lack of

evidence of an association, since there was no statistical difference (P>0.05).

However, the G variant of the 1911A>G polymorphism in TRPV1 was more frequent

in patients than in controls (42.8%) than in the controls group (36.0%, OR=1.28, 95%

CI 1.00-1.65, P=0.04) (Table 5).

In a second approach, the linkage disequilibrium (LD) between each pair in TRPV1 as

well as TRPA1 was checked by using SHEsis software. Two polymorphisms (2235

C>T and A787T) in TRPM 8 were not included into the haplotype calculation due to

the low frequency. All the P-value corresponding to haplotypes are shown in Table 6.

There was significant difference between patients and controls groups in the

haplotype C-A consisting of TRPV1 1103 T>C and 1911A>G. The frequency of

haplotype C-A was lower in patients (46.1%) than in controls

(53.8%, ,OR=0.73,95%CI 0.57-0.94) (p=0.02). The global haplotype frequency,

- 25 -

however, showed no significant differences between patients and controls in TRPA1

(χ2=6.44, P=0.09), TRPV1 (χ2=1.62, P=0.65) and TRPM8 (χ2=0.67, P=0.72).

Tab. 5: Allele frequencies and frequencies of genotypes in 296 patients and in 253 volunteers

Gene Location SNP

Allele Allele Frequency (%) p-value Odds ratio Geno- type

Frequency of genotype(%)

p

mRNA Protein Pat Controls d.f.=1 (95%)CI Pat Controls d.f.=2 TRPV1 G1103C Met315Ile C 75.0 72.8 0.4 1.122 CC 56.6 52.4 0.63 G 25.0 27.2 (0.85-1.48) CG 36.8 40.7 GG 6.6 6.9 A1911G Ile585Val A 63.2 57.2 0.04 1.285 AA 41.0 33.2 0.14 G 36.8 42.8 (1.00-1.65) AG 44.4 48 GG 14.6 18.8 TRPM8 C2235T Thr762Ile C 100 99.8 0.93 - CC 100 99.6 0.93 T 0.0 0.2 CT 0.0 0.4 TT 0.0 0.0 G1296A Ser419Asn G 93.6 94.9 0.48 0.83 GG 87.5 91.1 0.43 A 6.4 5.1 (0.49-1.38) GA 12.2 7.6 AA 0.3 1.3 G780C Arg247Thr G 94.2 95.8 0.22 0.42 GG 88.3 92.4 0.05 C 5.8 4.2 (0.81-2.47) GC 11.7 6.8 CC 0.0 0.8 A787T Pro249Pro A 99.5 99.4 0.82 1.17 AA 99.0 98.8 0.82 T 0.5 0.6 (0.19-7.26) AT 1.0 1.2 TT 0.0 0.0 G790C Leu250Leu G 88.9 90.4 0.41 1.17 GG 77.7 81.2 0.31 C 11.1 9.6 (0.79-1.75) GC 22.3 18.4 CC 0.0 0.4 A792G Tyr251Cys A 94.3 95.6 0.35 0.77 AA 88.7 92 0.08 G 5.7 4.4 (0.44-1.38) GA 11.3 7.2 GG 0.0 0.8 TRPA1 C182T Arg3Cys C 86.0 85.0 0.66 1.08 CC 70.1 72.3 0.54 T 14.0 15.0 (0.76-1.52) CT 29.9 27.3 TT 0.0 0.0 G710A Glu179Lys G 89.0 89.4 0.83 1.04 GG 79.7 80.4 0.98 A 11.0 10.6 (0.70-1.53) GA 18.6 18.0 AA 1.7 1.6 A3228G His1018Arg A 83.3 83.7 0.84 0.97 AA 70.0 69.1 0.36 G 16.7 16.3 (0.70-1.33) AG 26.6 29.3 GG 3.4 1.6 P-value of genotype was calculated by using wild-type homozygotes vs. heterozygotes and variant homozygotes

- 26 -

Tab. 6: Estimated haplotype frequencies in 296 patients and 253 controls TRPV1 G1103C A1911G Frequency (%) P-value OR (95% CI) Global result

haplotype Pat Control

1 C A 53.6 46.1 0.02 1.35(1.06-1.73) χ2=6.44 2 C G 21.4 26.7 0.05 0.75(0.56-1.00) df=3 3 G A 9.6 11 0.44 0.85(0.57-1.28) P=0.09 4 G G 15.4 16.2 0.74 0.94(0.67-1.32)

TRPA1 C182T G710A A3228G Frequency (%) P-value OR (95% CI) haplotype Pat Control

1 C A A 0.4 1.3 - - χ2=0.98 2 C A G 10 9.2 0.67 1.10(0.72-1.66) df=3 3 C G A 75 73.6 0.92 1.01(0.76-1.35) P=0.81 4 C G G 0.4 1.4 - - 5 T A A 0 0 - - 6 T A G 0 0 - - 7 T G A 9.1 7.7 0.38 0.82(0.53-1.27) 8 T G G 5.9 5.4 0.74 1.09(0.64-1.85)

TRPM8 G1296A G780C G790C A792G Frequency (%) P-value OR (95% CI) haplotype

1 G G G A 82.9 84.7 0.44 0.88(0.62-1.23) χ2=0.67 2 G G C A 10.4 9.6 0.66 1.10(0.73-1.64) df=2 3 A C G G 5 4.2 0.51 1.21(0.68-2.15) P=0.72 4 G C G G 0.2 - - - 5 G C G A 0.2 - - - 6 G G G G - 0.2 - - 7 A C C G 0.6 - - - 8 A G C A 0.1 - - - 9 A G G A 0.6 1.2 - -

The estimated haplotype frequency of patients and controls was compared by using SHEsis software platform.

3.3. Impact of polymorphisms on pain perception

To explore the association between selected SNPs and 13 QST parameters,

Kruskal-Wallis test and Jonckheere-Terpstra test were performed to test for

subgroups. The results are presented from Table 7 to Table 12. There was a clear

correlation between TRPA1 polymorphisms 710G>A and MPT and MPS,

as well as between 3228A>G and MPT and MPS of test side in central pain subgroup

- 27 -

(710G>A, P=0.021 and P=0.021, respectively, Kruskal wallis test; 3228A>G, P=0.007

and P=0.007, respectively, Kruskal wallis test, as shown in

Table 6).The homozygote A-allele carrier in 710G>A showed less sensitivity to pain

perception than GA allele at locus 710 in mechanical pain sensitivity (MPS)

measurement (P=0.028 Mann-Whitney U test) (Figure 6a). Moreover, in machincal

pain thresholds (MPT) measurement, the heterozygote 710GA seems less sensitive

to pain perception than homozygote G allele carrier (Figure 6b). The MPT z-score of

test side in TRPA1 polymorphism A3228G indicated that the homozygote 3228G

carriers were more likely to be loss of sensitivity than homozygote 3228A and

heterozygote GA carriers (P=0.032 and P=0.048, respectively (Figure 6C). Similarly,

the comparison of MPS z-score of test side apparently showed that the TRPA1

homozygote 3228G carriers were also more insensitive to the pain perception than

homozygote 3228A but not heterozygote carriers( P=0.03 Mann-Whitney U test ).

In CRPS patients, statistically significant associations were found between the

TRPV1 polymorphism 1911A>G and MPS on test side (P=0.003). The homozygote

1911G carrier showed less sensitive to pain perception than homozygote 1911A

(P=0.011) (Figure 6e). Compared the cold detection threshold (CDT) and thermal

sensitivity limen (TSL) with each SNP, there was also markedly significant difference

in TRPA1 polymorphisms 1911A>G (P=0.03 and P=0.046, respectively, Kruskal

wallis test) on control side, the further comparison depending on the genotypes

indicating patients carrying homozygote 3228G was more sensitive to cold stimui than

heterozygote 3228AG and homozygote 3228A carriers (P=0.026 and P= 0.024,

respectively, Mann-Whitney U test) (Figure 6g).

- 28 -

Tab. 7: Association between pain perception and SNPs from 3 TRP genes in 17

central pain patients.

CDT WDT TSL CPT HPT MDT T C T C T C T C T C T C

Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G780C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A787T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A792G a a a a a a a a a a a a TRPA1 C182T a a a a a a a a a a a a G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s MPT MPS WUR VDT PPT T C T C T C T C T C Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G780C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A787T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A792G a a a a a a a a a a TRPA1 C182T a a a a a a a a a a

G710A 0.021 0.043 0.021 n.s n.s

(K-W) (K-W) (K-W) n.s n.s n.s n.s n.s

0.004 0.004

(J-T) (J-T)

A3228G 0.007 n.s 0.007 n.s n.s

(K-W). (K-W) n.s n.s n.s n.s n.s

0.001 0.002

(J-T) (J-T)

K: Kruskal-Wallis test; J: Jonckheere-Terpstra test; T: test side; C: control side; n.s : no significance a .The Kruskal Wallis Test was conducted to compare 13 items of QST parameters with selected SNPs from TRP gene. b: The Kruskal wallis test cannot be performed in “a” row due to lack of valid cases. In this subgroup, there is a significant correlation between TRPA1 polymorphisms 710G>A and MPT and MPS, the significant correlation between TRPA1 A3228G and MPT and MPS. Furthermore, the Jonckheere-Terpstra test on test side suggests there is a z-score trend dependent on genotypes.

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Tab. 8: Association between pain perception and SNPs from 3 TRP genes in 56 CRPS patients

CDT WDT TSL CPT HPT MDT T C T C T C T C T C T C

Genes SNPs TRPV1 G1103C

n.s 0.03(K) n.s 0.045(J)

n.s n.s n.s n.s n.s n.s n.s n.s n.s

A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G780C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A787T a a a a a a a a a a a a G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A792G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPA1 C182T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

G710A n.s 0.035(K)n.s

0.014(J) n.s n.s n.s n.s n.s n.s n.s n.s n.s

A3228G n.s 0.03(k) n.s

0.022(J) n.s n.s 0.03(K) n.s

0.022(J) n.s n.s 0.016(K) n.s n.s

MPT MPS WUR VDT PPT T C T C T C T C T C Genes SNPs

TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

A1911G n.s n.s 0.003(K) n.s

0.001(J) n.s n.s n.s n.s n.s n.s

TRPM8 C2235T a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G780C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A787T a a a a a a a a a a G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A792G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPA1 C182T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

A3228G n.s n.s n.s n.s 0.019(K) n.s

n.s n.s n.s n.s

K: Kruskal-Wallis test; J: Jonckheere-Terpstra test; T: test side; C: control side; n.s : no significance In CRPS patients, there is a significant correlation was found between CPT of control side and TRPV1 G1103C, TRPA1 G710A and A3228G, there are also statistically significant correlation between TSL of control side and TRPA1 A3228G, MPS of test side and TRPV1A1911G. Furthermore, Jonckheere-Terpstra test shows significant trends in data mentioned above. However, the significant trends were not found between HPT of control side and TRPA1 A3228G, WUR of test side and TRPA1 A3228G.

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Table 9: Association between pain perception and SNPs from 3 TRP genes in 27 peripheral nerve injury patients

CDT WDT TSL CPT HPT MDT T C T C T C T C T C T C

Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s 0.008(k) n.s n.s 0.024(k) n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a a a

G1296A n.s n.s n.s n.s n.s n.s 0.035(k) n.s

0.033(J) n.s n.s n.s n.s

G780C n.s n.s n.s n.s n.s n.s 0.033(k) n.s

0.033(J) n.s n.s n.s n.s

A787T a a a a a a a a a a a a G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A792G a a a a a a a a a a a a

TRPA1 C182T 0.04(k)0.024(k) n.s

0.004(J)0.024(J) n.s n.s n.s 0.005(k) n.s

0.005(J) n.s n.s n.s n.s

G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s MPT MPS WUR VDT PPT T C T C T C T C T C Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G780C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A787T a a a a a a a a a a G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A792G a a a a a a a a a a

TRPA1 C182T 0.027(k) n.s

0.027(J) n.s n.s n.s n.s n.s n.s n.s n.s G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s K: Kruskal-Wallis test; J: Jonckheere-Terpstra test; T: test side; C: control side; n.s : no significance In peripheral nerve injury patients, z-score of CPT of test side is significantly associated with TRPM8 G1296A, G780C and TRPA1 C182T. There is significant correlation between CDT of test side and TRPA1 C182T. Similarly, the z-score of MPT of test side is significantly associated with TRPA1 C182T. Morover, Jonckheere-Terpstra test shows significant trends in data mentioned above , but there is no significant trends comparing TRPV1A1911G with WDT and CPT.

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Table 10: Association between pain perception and SNPs from 3 TRP genes in 27 PHN patients

CDT WDT TSL CPT HPT MDT T C T C T C T C T C T C

Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s 0.024(k) n.s n.s n.s n.s n.s

G780C n.s n.s n.s n.s n.s n.s 0.021(k) n.s

0.021(J) n.s n.s n.s n.s

A787T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

A792G n.s n.s n.s n.s n.s n.s 0.021(k)n.s

0.021(J) n.s

n.s n.s n.s

TRPA1 C182T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

G710A n.s n.s n.s n.s n.s n.s 0.044(k) n.s

0.044(J) n.s

n.s 0.049(k) n.s

0.049(J) A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s MPT MPS WUR VDT PPT T C T C T C T C T C Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s 0.037(k) n.s n.s n.s

G780C n.s n.s n.s n.s n.s n.s 0.029(k) n.s

0.029 (J) n.s n.s

A787T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

A792G n.s n.s n.s n.s n.s n.s 0.029(k) n.s

0.029(J) n.s n.s

TRPA1 C182T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

G710A 0.031(k) n.s

0.031(J) n.s n.s n.s n.s n.s n.s n.s n.s

A3228G 0.022(k) n.s

0.022(J) n.s n.s n.s n.s n.s n.s n.s n.s

K: Kruskal-Wallis test; J: Jonckheere-Terpstra test; T: test side; C: control side; n.s : no significance In post-herpetic neuralgia patients, z-score of CPT of test side is significantly associated with TRPM8 G1296A, G780C and TRPA1 G710A. A significant correlation was found between VDT of test side and TRPM8 G780C as well as A792G. Meanwhile, the z-score of MPT of test side is significantly associated with TRPA1 G710A as well as A3228G. Additinally, there is a significant correlation between MDT of test side and TRPA1 G710A. Morover, Jonckheere-Terpstra test shows significant trends in data mentioned above. The significant trend was not observed between CPT of test side and TRPM8 G1296A.

- 32 -

Table 11: Association between pain perception and SNPs from 3 TRP genes in 71 polyneuropathy patients

CDT WDT TSL CPT HPT MDT T C T C T C T C T C T C

Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s 0.037(k) n.s n.s n.s n.s 0.029(k) n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G780C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A787T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A792G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPA1 C182T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s MPT MPS WUR VDT PPT T C T C T C T C T C Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

G780C n.s n.s n.s 0.041(k) n.s

0.041(J) n.s n.s n.s n.s n.s

A787T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G790C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

A792G n.s n.s n.s 0.045(k) n.s

0.045(J) n.s n.s n.s n.s n.s

TRPA1 C182T n.s n.s n.s n.s 0.045(k)0.045(k) n.s

0.045(J)0.045(J) n.s n.s n.s

G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s

K: Kruskal-Wallis test; J: Jonckheere-Terpstra test; T: test side; C: control side; n.s : no significance In polyneuropathy patients, the MPS of test side is significantly associated with TRPM8 G780C as well as A792G. A significant correlation was found between WUR of both sides and TRPA1 G780C.Furthermore, further Jonckheere-Terpstra test suggests significant trends in data mentioned above. There is significant association without trend between TRPV1 G1103C and TSL of control side as well as MDT of test side

- 33 -

Tab. 12: Association between pain perception and SNPs from 3 TRP genes in 30 trigeminal patients

CDT WDT TSL CPT HPT MDT T C T C T C T C T C T C

Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G 0.035(k) n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a a a G1296A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G780C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A787T a a a a a a a a a a a a

G790C n.s n.s n.s n.s 0.024(k) n.s

0.024(J) n.s n.s n.s n.s n.s n.s

A792G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPA1 C182T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s MPT MPS WUR VDT PPT T C T C T C T C T C Genes SNPs TRPV1 G1103C n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A1911G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s TRPM8 C2235T a a a a a a a a a a

G1296A n.s n.s n.s n.s n.s n.s 0.041(k) n.s

0.041(J) n.s n.s

G780C n.s n.s n.s n.s n.s n.s 0.04(k) n.s

0.04(J) n.s n.s

A787T a a a a a a a a a a

G790C n.s n.s 0.008(k)0.012(k)n.s

0.008(J)0.012(J) n.s n.s n.s n.s n.s

A792G n.s n.s n.s n.s n.s n.s 0.04(k) n.s

0.04(J) n.s n.s

TRPA1 C182T n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s G710A n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s A3228G n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s K-W:Kruskal-Wallis test;J-T:Jonckheere-Terpstra test; T: test side; C: control side; n.s : no significance In trigeminal patients, the TSL of test side is significantly associated with TRPM8 G790C. A significant correlation was found between MPS of both sides and TRPM8 G790C. Similarly, there is significant association between the VDT of test side and TRPM8 G1296A, G780C as well as A792G. Furthermore, further Jonckheere-Terpstra test suggests significant trends in data mentioned above. There is significant association but trend between TRPV1 A1911G and CDT of test side.

- 34 -

Considering the TSL, the homozygote G-allele carriers in TRPA1 SNP 3288A>G

seems also less tolerance to thermal stimuli than homozygote carriers 3288 A

(P=0.045) (Figure 6h). However, there was no significant influence on pain perception

dependent on genotypes in the other SNPs compared with QST parameters.

(a)

(b)

Figure 6a: Comparison of MPS of test side depending on TRPA1 G710A genotypes in central pain patients

Figure 6b: Comparison of MPT of test side depending on TRPA1 G710A genotypes in central pain patients

- 35 -

(c)

(d)

(e)

Figure 6c: Comparison of MPT of test side depending on TRPA1 A3228G genotypes in central pain patients

Figure 6d: Comparison of MPS of test side depending on TRPA1 A3228G genotypes in central pain patients

Figure 6e: Comparison of MPS of test side depending on TRPV1 A1911G genotypes in complex regional pain symdrom (CRPS) patients

- 36 -

Figure 6: Comparison of z-score of QST parameters in some associated SNPs dependent on genotypes. (P-values calculated by Mann-Whitney U-test.)

(f)

(g)

(h)

Figure 6f: Comparison of CDT of control side depending on TRPV1 G1033C genotypes in centrol pain patients

Figure 6g: Comparison of CDT of control side depending on TRPA1 A3228G genotypes in GRPS patients

Figure 6h: Comparison of TSL of control side depending on TRPA1 A3228G genotypes in GRPS patients

- 37 -

4. DISCUSSION

4.1. The characteristics of neuropathic pain in the seven subgroups

Neuropathic pain is a chronic pain syndrome that has been associated with drug-,

disease-, or injury-induced damage or destruction of the sensory afferent fibers of the

peripheral nervous system (PNS), and has a substantial impact on quality of life and

mood [30]. Our clinical data, which include seven types of neuropathic pain, showed

that sensory alternations diversified depending on these different diseases. For

example, in central pain patient, post-herpetic neuralgia PHN as well as

polyneuropathy patient, the pain patterns characterized by loss of sensory function

are apparently distinct from the complex regional pain syndrome type (CRPS) patients,

who often exhibited the pain pattern of gain of sensory of function like hyperalgesia.

Our findings are consistent with the phenomenon in previously published literature, in

which the type of pain could manifest not only with positive sensory phenomena, such

as pain, dysesthesia, and different types of hyperalgesia, but also with negative

sensory phenomena and negative and positive motor symptoms and signs [31, 32].

Among thirteen QST parameters applied, the z-score of cold detection threshold

(CDT), mechanical detection threshold (MDT), thermal sensitivity limen (TSL),

mechanical detection threshold (MDT) and vibration detection threshold (VDT) in

central pain showed negative alterations in sensory function, suggesting that small

and large sensory fiber like afferent C- or Aδ-fiber could be implicated as a

consequence of long-duration pathological lesion. More interestingly, the VDT z-score

of CRPS patients showed hyperalgesia to blunt pressure, a phenomenon of feeling

vibration response to in-noxious stimuli. In contrast, the VDT z-score in peripheral

nerve injury and polyneuropathy subgroups exhibited the loss of sensory function.

Similarly, the MDT z-score of PHN and polyneuropathy patient indicated loss of

function in mechanical stimuli sensitivity. It is reported that Aβ-fiber is represented by

the VDT and MDT, May be lesions involving this type of afferent fiber probably result

in the gain or loss of function of mechanical sensitivity. Since the ability of peripheral

nervous system (PNS) to sense and perceive pain is an essential protective

- 38 -

mechanism that warns against threatened or ongoing tissue damage. Disruption of

this protective mechanism may result in the development of chronic pain. On the other

hand, the lower efficiency of endogenous pain modulation induced by alternation in

CNS pain processing, which in part attenuate the pain inhabitation, probably play a

crucial role in the origin of the neuropathic pain. In addition, several molecular

mechanisms, such as abnormal release of neurotransmitter (e.g. norepinephrine, NE),

increased neuronal hyperexcitability as well as unbalanced between excitatory (e.g.

glutamate) and inhibitory (e.g gamma-aminobuytric acid, GABA) transmitter system,

commonly lead to the dysfunction on pain signalling pathway, and creating a state of

allodynia and hyperalgesia or hypoesthesia [33, 34].

4.2 Neuropathic pain and single nucleotide polymorphism

Due to the multifactorial aetiology of neuropathic pain, a variety of treatment

algorithms were adopted by clinicians to alleviate the symptoms. But the overall

successful rate for treatment seems low because of the universal individual variability

and needs to be improved urgently. Meanwhile, this intriguing phenomenon draws lots

of attention to explore the underlying mechanism for relevant genes or SNPs, which is

recently suspected to play a crucial role in the variability of pain perception. Up to now,

there have been few genetic studies on human suggesting that single nucleotide

polymorphisms of specific genes could contribute significantly to basal pain sensitivity.

For example, Single-nucleotide polymorphisms of the µ-opioid receptor gene like

OPRM1 and the catechol-O-methyltransferase (COMT) gene were shown to be

associated with pain ratings in response to standardized noxious stimuli and with

pain-induced µ-opioid receptor binding in the CNS [22, 35, 36]. The polymorphism

OPRM1A118G, however, leading to a lower affinity of ligands to the extracellular

binding domain of the µ-opioid-receptor, seems more likely to be associated with the

acute pain [37]. It is commonly reported that postoperative patients carrying

homozygously 118G, exhibit a higher pain sensitivity and require higher opioids

dosages than those being homozygous for 118A. Another common functional

polymorphism 1947G>A in COMT gene cause substitution from valine to methionine

- 39 -

at amino acid position 158, which is suggested to increase pain sensitivity and to

decrease opioid system activation in the brain [38]. Haplotypes including COMT

Val158Met were recently identified and an association was suggested with

experimental pain sensitivity and a chronic pain condition [22]. However, there are still

controversial findings concerning this SNP in acute pain patients or in different

ethnicities [15, 33].

Recently, there is increasing evidence supporting that hypersensitivity and pain that

occurs under various pathological conditions is often due to up-regulated expression

and/or increased sensitivity of TRP channels. Several lines of evidence point to the

involvement of members of the TRPV family in the aetiology of neuropathic pain

[39,40], So far, considering the potentially functional role of polymorphism in TRP

gene, only few information on humans has been explored regarding the association

between pain perception and TRP genetic polymorphisms. Recently, a study led by

Xu et al [41] suggested that TRPV1 Met315lle variant might lead to functional

difference at the level of expression at the cell surface, on a HEK293 cells model.

Furthermore, a body of previous literature focuses on racial differences in the pain

perception response to painful stimuli [42]. For example, two clinical studies found

that Asian patients may require less post-operative analgesia than European patients

[43]. This phenomenon, however, has not been replicated in all reported studies [44].

4.3 Genetic variability of TRPV1 and their influence on pain perception

In this study, the genotype distribution of 11 selected TRP gene polymorphisms were

examined in neuropathic pain patients and volunteers, trying to explore whether there

has been a biologically relationship between these SNP and alterations of pain

sensitivity. Among these markers, two common single nucleotide polymorphisms

TRPV1 1103 C>G and 1911 A>G, also known as TRPV1 Met315Lle and LIe585Val,

result in substitution from methionine to isoleucine at codon 315 and isoleucine to

valine at codon 585,repectively[22, 45]. They are 12.7 kb apart from each other in the

genomic sequence. We found the allelic frequency for the 1911 A>G polymorphism

was statistically significant higher in patients compared with controls (P=0.04). The

- 40 -

overall G allelic frequency in patients was 36.8%, which was similar to those reported

in the HapMap database (34.2%) and in the study led Kim et al (36.0%). More

interestingly, the overall allelic frequency of the 1911A>G polymorphism in control

group approaches 42.8%. Although the genotype frequency of 1911GG carriers in

control group was 18.8%, slightly higher than the patient group (14.6%), there is no

significant difference (P=0.14, χ2=3.86) between patient and control group. Our data

still indicated that neuropathic pain patients were less likely to carry the TRPV1

1911G allele at this position.

Until now, conflicting findings over TRPV1 polymorphisms still exist. For the TRPV1

1911A>G polymorphism, no different functional response to capsaicin, pH and

temperature between the different alleles has been detected in whole-cell

patch-clamp and calcium imaging experiments, suggesting that the substitution does

not critically alter receptor structure or function, at least in vitro[46]. Conversely, Kim et

al [47] observed that this polymorphism alters cold withdrawal times (CWT i.e,

increased sensitivity to cold-induced pain). From several animal experiments, there

was some evidence derived supporting that TRPV1 after long-term activation could

exert a protective role in the development of mechanical hyperalgesia [48], even

though the detailed mechanism was not finally clarified. Considering our control group

with the higher allele frequency in TRPV1 1911A>G, the question remains open as to

whether the carrier with G allele in neuropathic pain subject have a potentially

protective role from aggravating into further stage. So, further study should be

investigated to confirm this hypothesis.

TRPV1 likely plays a major role in integrating nociceptive signals, particularly in the

contexts of inflammatory and neuropathic pain [49, 50]. When comparing genotype

data in patients with 13 of the PSQ parameters, a statistically significant correlation

was found between mechanical pain sensitivity (MPS), cold detection threshold (CDT)

and polymorphism TRPV1 1911A>G in CRPS patients (P=0.003 and

P=0.03,respectively). Our results are not consistent with the previously published

finding by Kim et al [22], in which they failed to find any significant association

between these two polymorphisms in TRPV1 and cold/heat pain sensitivity in

- 41 -

American female subjects of European descent. In their study, the different

experimental environment used, which includes taking healthy subjects, different

methods of pain measurement, pain parameters selected as well as the racial

difference may explain this inconsistency. In our study, MPS was designed to detect

an typical phenomenon, termed pinprick hypoesthesia, which was often complicated

with the neuropathic pain and was believed to be consistent with a large fiber sensory

deafferentation like afferent Aδ-fiber, and the presence of abnormal cold pain

threshold (CDT) in GRPS subgroup also indicates a disturbance of Aδ-cold sensory

function, which might indirectly reflect the damage extent on the afferent pain

signalling pathway, and evaluate the influence on the pain sensitivity. However, the

relative contribution of C- and Aδ-fiber nociceptors to cold and pressure pain

threshold is less clear [28, 51]. Based on our results, we could conclude that TRPV1

1103G>C and 1911A>G was closely associated with the pain perception in certain

type of neuopathic pain, especially for thermal and mechanical sensitivity. Many

evidences suggested TRPV1 plays an important role in chemical and thermal

hyperalgesia in a model of diabetic neuropathy [52]. Its role may be associated with

decreased cell-specifically TRPV1 protein expression in C-fibers paralleled by an

increase in A-fibers, leading to an increase in its function. Furthermore, a possible

“cooperation” between TRPA1 and TRPV1 channels has also been reported in recent

vitro study, in which the canabinoid agonist WIN 55,212-2 dephosphorylates,

therefore desensitizing TRPV1 in trigeminal sensory neurons via the activation of

TRPA1.

With regard to the comparison of the MPS z-value with TRPV1 polymorphisms, the

results of test side clearly showed that homozygote 1911G carriers are more likely to

exhibit the loss of sensory function than homozygote A1911 carriers. Similarly, the

comparison of mechanical pain threshod (MPT) z-score on control side indicated that

subjects carrying TRPV1 1911GA are less sensitive to the cold stimuli than those who

carrying homozygote 1911A, representing loss of sensory function. Impaired pain

sensation might result from distorted TRPV1 regulation in the peripheral nervous

system, there is quite a little vivo data recently supported that TRPV1 protein

- 42 -

expression could be enhanced by IGF-I via PI(3)K signal pathway [53]. Moreover, as

a target of the cAMP/PKA signal pathway, the effect of TRPV1 under tissue

inflammation or nerve injury might also be influenced by alteration of PKA-mediated

phosphorylation [54].So, it can also be speculated that the substitution of amino acid

caused by SNP TRPV1 1103G>C and 1911A>G probably alters biological function on

afferent fiber or primary sensory neuron by altering secondary structure of mRNA or

TRPV1 protein expression, leading to individual differences of pain sensitivity.

4.4 Association of TRPM8 genotypes with neuropathic pain

We found no difference in the genotype or allelic frequencies for these five SNPs in

TRPM8 gene between patients or controls, indicating that there was no evidence for

an association of risk of developing neuropathic pain. The genotype or allelic

frequencies in our patients and controls were also similar to those reported in HapMap

database. Interestingly, some SNPs in the TRPM8 gene, namely 2235C>T (Thr762Tle),

780G>C (Arg247Thr), 787A>T (synonymous), 790G>C (synonymous) and 792A>G

(Tyr251Cys), could not be found in our patients. Among these SNPs, two out of them

do not seem to be polymorphic in our volunteers. From the HapMap, we found that the

minor allele frequency of 780 A>C and 790G>C in Asia population but not in

European population was always polymorphic, indicating the existence of an ethnic

difference in these two SNPs. TRPM8 is a non-selective, outwardly rectifying channel

that can be activated by cold (8-28 °C) [55], Up to date, there is increasing evidence

that mutation in coding regions could contribute to the function of the TRPM8 channel.

Dragoni et al [56] have identified two potential N-glycosylation sites in TRPM8

(Asn-821 and Asn-934) in rats, and observed the decreased efficiency of synthesis of

mature, glycosylated TRPM8 as a result of mutation. It is widely accepted now that

TRPM8 may play a major role in certain types of cold-induced pain [57].

In this study, comparing five TRPM8 SNPs with 13 QST parameters, no significant

association was found dependent on the genotypes mostly because of no valid case

in each subgroup. Also Kim et al failed to find any association between TRPM8

variant and cold as well as heat pain sensitivity in their healthy European Americans.

- 43 -

However, considering the relatively higher range of threshold temperature to TRPM8

(8-28 °C), it is likely that the TRPM8 receptor was not activated by the painful cold

temperature they used (2-4 °C) [22]. Nevertheless, it still raises the intriguing

possibility that genetic polymorphisms, such as those investigated here, may account

for or contribute to a racial difference in pain threshold or tolerance. In addition, animal

experiments showed that mutation in TRPM8 gene could contribute to the function of

this TRPM8 channel [57]. It is possible, therefore, that one SNP may be in linkage with

another functional SNP in the TRPM8 gene, inducing an impact on behavioural

phenotype such as pain perception difference through an epigenetic mechanism

related to this nucleotide substitution.

Duan et al [58] showed that synonymous SNPs within haplotypes may have functional

consequences drastically different from those of each isolated variant. This effect was

attributed to alterations in the secondary structure of mRNA, which result in weakened

mRNA stability or protein translation. Furthermore, it has been well documented that

TRPM8 is mostly present in small diameter sensory neurons, and all

TRPM8-expressing neurons also expressed receptor tyrosine kinases (TrkA), which is

a NGF receptor [59]. For those TRPM8 SNPs involving amino acid substitution, such

as 1296G>A and 780G>C, the question remains open whether these polymorphisms

might alter the expression of TRPM-8 receptor through the nerve growth factor (NGF)

pathway.

4.5 Genetic variability of TRPA1 and association to pain perception

The genotype and allele distribution of three TRPA1 SNPs investigated was identical

in our patients and controls, and there was no evidence for an association to risk of

neuropathic pain. All the genotype and allelic frequencies were similar to those

reported in Hapmap database in European population supporting the generalizability

of the findings. Regardless of TRPA1 182C>T, the minor allelic and genotype

frequency of TRPA1 710G>A and 3228A>G reported in HapMap database showed

significant difference among ethnic groups, which should also be considered in

association studies with SNPs and phenotypes.

- 44 -

Considering the results of pain perception in central pain subgroup, we found that

TRPA1 SNPs 710G>A and 3228A>G were significantly associated with MPT and

MPS on affected side of the patient. Compared mechanical pain threshold (MPT)

z-score depending on the genotype in SNPs A3228G, central pain patients carrying

homozygously the 3288G allele, tended to be insensitive to pain perception than

homozygote 3288A carriers (P=0.032 in MPT and P=0.03 in MPS, respectively,

Mann-Whitney U-test) and heterozygote GA carriers (P=0.048 in MPT). A similar

result was found for TRPA1 710G>A, but possibly due to the low allelic frequency - the

difference was only significant in heterozygote GA carriers (P=0.013 in MPT and

P=0.013 in MPS, respectively, Mann-Whitney U-test). The decreased mechanical

pain thresholds or pain sensitivity in central pain patients could be linked to the

disturbance of afferent Aδ-fiber. Furthermore, the MPT or MPS parameter could be

used to detect the mechanical pain stimuli and to determine whether patients with

pain processing abnormalities (i.e. neuropathic pain) show exaggerated temporal

summation of second pain intensity [49]. The mechanism involving

N-methyl-D-aspartate (NMDA) receptor within the dorsal horn is thought to be

responsible for temporal summation of pain [60]. This finding was partially in

agreement with the receptor function in previously published literature [61], in which

the TRPA1 was believed to be a sensor for mechanical stimuli. Under neuropathic

pain status, alterations in neuronal function, chemistry, and structure, which often

occurred secondary to nerve injury, might result in abnormal expression of TRPA1

receptor, leading to loss of sensory function. It is still unclear which mechanism gets

involved in the decreased afferent fibers function in the homozygote 710A. The amino

acid substitution caused by this polymorphism from histidine to arginine at position

1018 could be one possible reason to contribute to this different sensitivity. In addition,

one interesting property of TRPA1 channels, which inactivate in hyperpolarized cells

but remain open in depolarized cells, also provides a mechanism for the lack for

desensitization, coincidence detection, and allodynia [63]. However, whether the

amino acid change induced by polymorphism alters these depolarized cells leading to

influence on iron influx still need to be considered in future studies.

- 45 -

The comparison data of QST parameters like thermal sensitivity limen (TSL) and cold

detection threshold (CDT) on the non-affected control side of the patient for TRPA1

A3228G also indicated that homozygote 3288G carriers of CRPS patients were more

sensitive to pain perception than homozygote A3288 (in CDT) and heterozygote AG

carriers (both CDT and TSL). A great amount of evidence supporting that TRPA1 has

also been linked to cold hyperalgesia behind neuropathic pain [38, 64]. Inflammation

and nerve injury increase the expression of TRPA1 in DRG neurons. Furthermore, the

co-expression of TRPA1 and TRPV1 and the flexibility of the TRP family channels

raise the possibility that TRPA1 channel might interact to influence the properties of

one another [19, 64]. Animal experiments showed that an NGF-induced TRPA1

increase in sensory neurons via p38 activation is necessary for cold hyperalgesia [38,

65]. Other studies further demonstrated that mice following trpa1 gene knock out or

trpa1 antagonist exhibited behavioural changes in response to the pain stimuli, which

illustrated the important role of TRPA1 in pain perception.

4.6 Limitation of our study

There are still some limitations in the study. Firstly, the volunteers were not

investigated by the QST test panel, which consequently failed to powerfully evaluate

the variability in pain perception among healthy subjects. Secondly, in the study,

patients in subgroups apparently showed different pain pattern dependent on the

aetiology of the neuropathic pain. Therefore we necessarily further compared z-score

with associated SNPs for each subgroup based on the genotype, which probably

increases the ability to study phenotype-genotype association. But owing to no valid

cases in some of subgroups, larger sample are necessary in order to get stronger

statistic power. Finally, functional test based on the vivo or vitro study should be

performed to directly measure the effect of these SNPs as to confirm the association

and to provide a stronger rationale for the relationship with pain sensitivity. Our

association analyses have identified several loci associated with thermal and

mechanical pain sensitivity, but their functional importance has yet to be confirmed.

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4.7 Conclusion

In conclusion, our results have a considerable clinical significance and represent the

first study of this kind which demonstrated an association between several genetic

polymorphisms like TRPV1 1103C>G, 1911A>G, 710G>A as well as 3228A>G and

pain sensitivity. These SNPs produce different influences on pain perception

dependent on the genotype in patients on the condition of neuropathic pain. As far as

complex regional pain syndrome (CRPS) patients are concerned, the subjects

carrying the minor homozygote 3228G exhibited more sensitivity in response to

thermal pain stimuli, than those with wild-type homozygote carriers. In contrast,

homozygote 1103C, 1911G carriers were more insensitive to mechanical and thermal

stimuli than homozygote G1103, A1911carriers, which were consistent with the

influence of homozygote 3288G carriers on WPT and WPS in central pain subgroup.

These polymorphisms exert major effects maybe by influencing the level of

expression of the gene product, dependent of its function. Finally, these

polymorphisms alone or interaction between SNPs may alter transcription, mRNA

stability, or the protein half-life, leading to the association with a certain clinical status.

For those polymorphisms without involving amino acid substitution, the observed

relationship between associated SNPs and pain perception could be caused by other

functional polymorphisms in the neighbouring genes that posses high LDs with tested

SNPs. Although there was no evidence of an association between genotypes and the

risk of being neuropathic pain, for the TRPV1 1911A>G, the relative lower proportion

in homozygote 1911G in patient compared to healthy volunteers possibly indicated

that subjects likely carrying the common G allele at this position have a potentially

protective role, preventing the aggravation in the further stages.

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5. SUMMARY

Neuropathic pain is a chronic pain syndrome that has been associated with drug-,

disease-, or injury-induced damage or destruction of the sensory afferent fibers of the

peripheral nervous system (PNS). The type of pain could be manifested not only with

positive sensory phenomena, such as pain, dysesthesia, and different types of

hyperalgesia, but also with negative sensory phenomena and negative and positive

motor symptoms and signs. The pharmacological treatment of the symptoms of

painful neuropathy, however, is still considered to be difficult. Nowadays, much

attention is focused on the genetic polymorphisms, some of the single nucleotide

polymorphisms can be speculated to be associated with the pain sensitivity as a result

of amino acid substitution in crucial position. But the detailed knowledge of individual

variability on pain perception under neuropathic pain is still poorly understood.

Candidate gene studies on the basis of biological hypothesis have been a practical

approach to identify relevant genetic variation in complex traits. There is growing

evidence showed that single nucleotide polymorphisms in related genes, including

TRPV1, TRPA1 and TRPM8, may influence pain sensitivity in animal model of

neuropathic pain.

In our study, we selected 2 of the SNPs fromTRPV1, 3 of the SNPs from TRPA1 and

6 SNPs from TRPM8 to examine the effect of these variations on clinical neuropathic

pain responses, to investigate the contribution of genetic factors on pain sensitivity in

humans. There were a total of 296 Germany patients and 253 healthy volunteers

recruited in our study for investigation. Owing to the failed collection of clinical data in

some cases, finally only 237 patients were enrolled to carry out the association

analysis. The results show that patients exhibited markedly gain of sensory function

along with the application of cold detection (CDT), thermal sensitivity limen (TSL) on

control side. The hypoesthesia, however, was occurred on test side when patients

were giving mechanical pain threshold (MPT) and mechanical pain sensitivity (MPS)

measure. The comparison of seven subgroups between test and control side showed

that there are different pain patterns based on the subgroups. Besides the trigeminal

pain and other neuropathy patients, the five other subgroups exhibiting either the loss

- 48 -

of sensory function in CDT, WDT, TSL and VDT on central pain, or the gain of

sensory function in pressure pain threshold (PPT) in CPRS patient. Referring to the

genotype and allele frequencies on patients and controls, there are no significant

differences between the two groups, except the TRPV1 polymorphism A1911G.

With regard to the association between SNPs and 13 QST parameters, there were

some significant correlations related to TRPV1 polymorphisms 1103 C>G and

1911A>G, as well as to TRPA1 polymorphism 710G>A and 3228A>G with several

QST parameters in two neuropathic pain subgroups. The CRPS patients carrying

homozygote G in TRPA1 3228A>G were more sensitive to pain perception than those

patients carrying the heterozygote genotype in the TSL and CDT test, conversely, in

some subgroups homozygote TRPV1 1911G carriers were more likely to be

insensitive to sensory pain than homozygote 1911A carriers; Similarly, further

comparison of the data indicated that patients carrying homozygote TRPA1 710A or

3228G were less sensitive to pain perception than heterozygote and homozygote

G710 or 3228A carriers.

The results suggest that at least some of these genetic polymorphisms may exert

major effects on pain perception possibly by influencing the level of expression of the

gene product, depending on its function. Finally, polymorphisms alone or interaction

between SNPs may alter transcription, mRNA stability, or the protein half-life, leading

to a specific clinical status. For those polymorphisms which do not involve amino acid

substitution, the observed association with pain perception could be caused by other

functional polymorphisms in the neighbouring genes that posses high LDs with tested

SNPs.

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7. Words of Thanks

I appreciated deeply Prof. Dr. Dr. Ingolf Cascorbi, Director of Institute experimental

and clinical Pharmacology, Kiel, for providing me the opportunity to perform this

important study and for his numerous efforts in helping me to achieve this work. I

sincerely thank Dr. Sierk Haenisch for his considerate and profound help in teaching

me the basic laboratories skills, particularly for his valuable statistical advice. The

skilful help of Dr. Lise-Marie Daufresne and Dr Yi Zhao are gratefully acknowledged. I

would like to thank Cornelia Remmler, Sandra Lächelt and Marion Pauer for their

kindness, gentleness and consideration to me during my study. I also send my thanks

to Dr. Adina Varelia Tocoian for her kindly help necessary to accomplish my study.

Finally, I like to thank the German Neuropathic Pain Network particularly Prof. Dr. Ralf

Baron, Department of Neurology, Kiel for providing me with the DNA samples and

valuable clinical data.

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CURRICULUM VITAE

Name: Zhu Jihong

Sex: Male

Place of birth: Zhejiang Province, P.R.China

Date of birth: Jan 30, 1974

Nationality: Chinese

Marital status: Married

Education:

1994-1998: Zhejiang University School of Medicine, P.R.China

(Bachelor Degree in Medicine)

2000-2003 Zhejiang University School of Medicine, P.R.China

(Master of Science)

Working experience:

1997-2002 Department of Anesthesia, Sir Run Run Shaw Hospital, Zhejiang

University School of Medicine, P.R.China (Resident Doctor in

Anesthesia)

2002-2003 Department of Anesthesia, Sir Run Run Shaw Hospital, Zhejiang

University School of Medicine, P.R.China (Research Fellowship)

2003-2006 Department of Anesthesia, Sir Run Run Shaw Hospital, Zhejiang

University School of Medicine, P.R.China (Clinical Anesthesia

Fellowship)

Jan.- Dec.2007 Institute of Pharmacology, University Hospital Schleswig-Holstein,

Campus Kiel, Kiel, Germany (Visiting scholar and research fellow)